RNA viruses replicate with extremely high mutation rates allowing them to quickly develop resistance to conventional drugs that directly target viral proteins. Viruses critically rely on cellular genes for their replication and spread. Therefore, using these host genes as drug targets could create a much higher barrier to the development of drug resistance. To identify host genes critical to infection of a wide range of different emerging or reemerging RNA viruses, we will use an innovative method for genetic screens in human cells. This approach is based on unique haploid or near-haploid human cell lines in which it is possible to create knockout alleles en masse creating a library of cells with mutations in essentially all genes. Coupled with deep sequencing this allows us to screen the human genome for genes that are critical for viral replication. We have applied this technique to identify entry factors required for Ebola virus and found that NPC1 is the intracellular receptor for different filoviruses. Here we propose to perform a series of comparative genetic screens for host genes required for a compendium of RNA viruses including different strains of influenza A virus, Venezuelan equine encephalitis virus. La Crosse encephalitis virus, yellow fever virus, West Nile virus, hepatitis A virus and hepatitis C virus. These screens will generate candidate genes that need to be rigorously validated in relevant tissue culture and animal models. For this we will develop AAV-shRNAs- RNAi tools. These will be used to: 1) validate host cell candidate genes identified and deemed important for the replication of viral pathogens; 2) compare efficacy of host target knockdown with direct targeting of viral mRNAs with host candidate genes; 3) identify the toxicity of small molecule inhibitors directly related to direct knockdown of the candidate protein from toxicity related to pleiotropic effects of the small molecule inhibitors.